The present invention relates to microfluidic devices with microelectrodes, more particularly to microfluidic devices, particularly to microfabrication technology for producing microchannels with embedded electrodes, and more particularly to a microfabricated AC impedance sensor for multi-frequency particle impedance characterization.
Microfabrication has become an enabling technology for development of the next generation of analytical instrumentation for performing medical diagnoses, sequencing the human genome, detecting air borne pathogens, and increasing throughput for combinatorial chemistry and drug discovery. These miniature devices take advantage of scaling laws and unique physical phenomena which occur at the micro-scale to perform new types of assays. The large surface area to volume ratio and small size of microfabricated fluidic devices results in laminar flows, increased surface contact between sample fluids and electrodes, fast and uniform heat transfer, and reduced reagent use. Surface tension and viscous forces dominate while inertial effects are negligible. Microfabricated electrodes can produce high electric fields for electroosmotic flows and electrophoretic separations, and large field gradients for inducing dielectrophoretic forces on particles.
The Coulter counter, a commercial instrument which uses DC impedance (resistance) measurements to determine the volume of small particles in suspension, has existed for over 40 years. Instruments that combine Coulter Principle and single high frequency impedance measurements to determine the cellular cytoplasmic conductivity are available for blood cell analysis. However, such two-parameter measurements are incapable of distinguishing between some particle subpopulations of interest (e.g., granulocytes and monocytes, or spores and background particles). What is desired for a host of hematology, pharmacology, forensic, and counter biological warfare applications is a system that can perform broad-band impedance characterizations of particles to enable superior particle differentiation via features in their impedance spectra. Recent research has shown that cellular parameters vary with such physiologic alterations as apoptosis, malarial infection, cell differentiation, and exposure to toxins.
A method often employed for broad-band electrical characterization of particles is electrorotation (ROT), whereby a rotating electric field is used to generate a torque on a particle. The magnitude and direction of the torque, and the resulting particle rotation, depend on the particle""s dielectric properties. By measuring a particle""s rotation rate as a function of excitation frequency, its conductivity, membrane capacitance, membrane resistance, and other properties can be determined. Although the technique is precise, it requires significant measurement time; even automated ROT systems take several minutes per particle.
A faster approach uses a pulse-FFT measurement scheme. As particles pass through a sensor orifice, a broadband electrical impulse is used to excite them. The particle""s impedance signature is then generated using the Fast Fourier Transform (FFT) of the sensor""s impulse response. This technique produces a quasi-continuous measure of the particle""s impedance spectra, but since the energy of the excitation signal is spread across a band of frequencies, the signal-to-noise ratio (SNR) is inherently worse than in a discrete frequency system.
Another approach involves the use of tuned oscillators to perform simultaneous impedance measurements at multiple, discrete frequencies which are chosen to capture the important transitions in the particle""s impedance spectra. Since the system""s signal power is concentrated at the measurement frequencies, this technique produces a better signal to noise ratio than the pulse-FFT approach. However, changes in the operating frequencies or solution conductivity may cause the oscillator circuit to become de-tuned. Such a system is most useful for measurements involving predetermined particle types under specific operating conditions.
Microfluidic devices with microelectrodes have the potential to enable studies of phenomena at size scales where behavior may be dominated by different mechanisms than at macroscales. Through work developing microfluidic devices for dielectrophoretic separation and sensing of cells and particles, we have fabricated devices from which general or more specialized research devices may be derived. Fluid channels from 80 xcexcm widexc3x9720 xcexcm deep to 1 mm widexc3x97200 xcexcm deep have been fabricated in glass, with lithographically patterned electrodes from 10 to 80 xcexcm wide on one or both sides of the channels and over topographies tens of microns in height. The devices are designed to easily interface to electronic and fluidic interconnect packages that permit reuse of devices, rather than one-time use, crude glue-based methods. Such devices may be useful for many applications of interest to the electrochemical and biological community.
The present invention involves a microfabricated flow-through impedance characterization system capable of performing AC, multi-frequency measurements on cells and other particles. The prototype sensor measured both the resistive and reactive impedance of passing particles, at rates of up to at least 100 particles per second. Its operational bandwidth approached 10 MHz with a signal-to-noise ratio of approximately 40 dB. The bandwidth on signal-to-noise ratio may be increased. Particle impedance is measured at three or more frequencies simultaneously, enabling the derivation of multiple particle parameters. This constitutes an improvement to the well-established technique of DC particle sizing via the Coulter Principle. Human peripheral blood granulocyte radius, membrane capacitance, and cytoplasmic conductivity were measured (r=4.1 xcexcm, Cmem=0.9 xcexcF/cm2, "sgr"int=0.66 S/m) and were found to be consistent with published values.
It is an object of the present invention to provide a microfabricated alternating current (AC) impedance sensor.
A further object of the invention is to provide microfluidic devices with microelectrodes to enable studies of phenomena at size scales where behavior may be dominated by different mechanisms than at macroscales.
A further object of the invention is to provide microfluidic devices with microelectrodes which are easy to interface to electronic and fluidic interconnect packages that permit reuse of devices, rather than one-time use, crude glue-based methods, useful for electrochemical and biological applications.
Another object of the invention is to provide a microfabricated flow-through impedance characterization system capable of performing AC, multi-frequency measurements on cells and other particles.
Another object of the invention is to provide a microfabricated AC impedance sensor which measures both the resistive and reactive impedance of passing particles, at rates of at least up to 100 particles per second, with an operational bandwidth which approaches at least 10 MHz with a signal-to-noise ratio of at least about 40 dB.
Another object of the invention is to provide a microfabricated AC impedance sensor wherein particle impedance is measured at three or more frequencies simultaneously, enabling the derivation of multiple particle parameters.
Other objects and advantages of the present invention will become apparent from the following description and accompanying drawings. The present invention involves a microfabricated instrument for detecting and identifying cells and other particles based on AC impedance measurements. Two critical elements of the invention are:
1. A glass or polymer-based microfluidic chip, fabricated using photolithographic and chemical etching techniques. Microchannels are etched into one glass substrate, and holes for introducing fluids and making electrical contacts are drilled into a second piece of glass. Platinum or gold electrodes are patterned on both glass substrates, using electrodeposited resist to create features at the bottom of the microchannels. The two pieces of glass are aligned and then fusion bonded using a vacuum hot press or anodically-bonded with an amorphous silicon intermediate layer. Microfabrication is essential for this application in order to make channels and electrodes that are small and precise enough for accurate measurements, and to allow the electrodes to be sufficiently localized to reduce fringing field effects. Using glass reduces parasitic capacitance problems associated with conductive materials such as silicon. Both of these enable multiple AC impedance measurements to be taken on individual particles at high throughput rates.
2. Electrical circuits that connect to the electrodes on the microfluidic chip and detect the signal associated with particles traveling down the microchannels. Two circuits are described: (1) A discrete circuit which relies on the difference in signal between a reference channel with solution only and a measurement channel through which both solution and particles travel. The discrete circuit is capable of measuring impedance changes associated with particles at one frequency, although more than one stage can be used, allowing for measurement at one additional frequency for each additional stage. (2) An integrated circuit which does not require a reference channel and is capable of measuring impedance changes at several frequencies simultaneously. Both of these circuits are capable of detecting a 1% impedance change within the microchannels to 1% accuracy. These circuits enable multiple AC impedance measurements of individual particles at high throughput rates with sufficient resolution to identify different particle and cell types as appropriate for environmental detection and clinical diagnostic applications.
Through work at the Lawrence Livermore National Laboratory developing microfluidic devices for dielectrophoretic separation and sensing of cells and particles, we have fabricated devices from which general or more specialized research devices may be derived. Fluid channels from 80 xcexcm widexc3x9720 xcexcm deep to 1 mm widexc3x97200 xcexcm deep have been fabricated in glass, with lithographically patterned electrodes from 10 to 80 xcexcm wide on one or both sides of the channels and over topographies tens of microns in height. The devices are designed to easily interface to electronic and fluidic interconnect packages that permit reuse of devices, rather than one-time use, crude glue-based methods.